1. Field of the Invention
The invention generally relates to the establishment of cell lines which are stably transfected with a detectable marker. In particular, the invention provides a DNA vector carrying the GFP marker which can be used to establish stably transfected cell lines; to use for monitoring cell movement, proliferation, and location in vivo; and the invention pertains to stably transfected cell lines which include the GFP marker.
2. Background of the Invention
The ability to transfect cells with exogenous DNA and to monitor such genetically engineered cells in vivo is of fundamental importance to many areas of scientific investigation and emerging medical treatments. For example, ex vivo gene therapy for several diseases depends on the ability to transfect cells. In the treatment of Parkinson's disease, it is possible to surgically remove neural progenitor cells from the patient, grow them in culture, insert therapeutic genes, and then replace the transfected cells back into the patient's brain. However, the ability to monitor correct cell placement and gene expression, which would be a valuable addition to this treatment, is currently not available. Similarly, there is much interest in developing new cancer diagnostic tools to monitor tumor cell invasion,
There are many ways to transfect cells with DNA. These include chemical transfections with lipids or lipid-type compounds, and shocking the cells by either chemical or electrical means. Another means of transfecting DNA into a cell is by using viral vectors (e.g. adenoviral and retroviral vectors) to transfect/transduce the host cells. However, with any of these techniques, the transfection of cells (whether mammalian, plant, or other type of cell) is usually transient, precluding long-term expression of the exogenous DNA or its gene products. For example, the use of viruses and dyes used to monitor cells are limited in that with subsequent cell divisions the ability of daughter cells to express the target DNA or dye diminishes with each cell division.
It would be highly desirable to have available a technique which would provide for the stable tranfection of cells. One important use of such stably transfected cells would be to monitor biological events in "real time". For example, it is currently not possible to detect the migration of cells such as brain tumor cells or neuronal stem cells. The provision of cells stably transformed with markers which allowed monitoring of biological events in real time would be highly advantageous.
One specific example of an area of medicine that would benefit from the ability to monitor real-time biological events is the field of brain tumor research, such as that involving glioblastoma multiforme, the most common primary brain tumor. Despite significant improvements in the diagnosis and treatment of patients with glioblastoma multiforme, it remains incurable. A key feature that underlies the malignant behavior of this disease is the ability of glioma cells to aggressively infiltrate surrounding brain tissue. Thus, understanding the basic biology of tumor cell invasion/migration may aid in the development of more effective forms of treatment [1-6].
Much information about glioma cell invasion has been gained from studies using a variety of in vivo and in viro models. Tumor cells have been labeled using Phaseolus vulgars leucoagglutinin (PHAL), cell labeling dyes, such as fast blue and transfection with the lacZ gene. Each technique has advantages and disadvantages [2, 7, 8 and references therein]. One disadvantage of using externally labeled cells is that long term studies (i.e. lasting multiple cell divisions) are difficult or impossible due to a decrease in signal over time. Although transfection with lacZ is stable, it requires post-processing using a chromogenic substrate for the .beta.-galactosidase marker enzyme. Caution must be exercised when introducing dyes and substrate reagents into cells because this treatment can affect biological processes such as cell proliferation and motility [9, 10], the very processes one would like to observe.
It would be a distinct advantage to have available an improved means to monitor cell motility in general, and tumor cell migration in particular, e.g. to easily and rapidly identify tumor cells that are in the process of migrating, and to enable examination of their biochemical properties.